Materials

Procedure

Preparing the 5X stock

Add the following reagents to a 2-liter flask:

5 g (NH4)2SO4

22.5 g KH2PO4

52.5 g K2HPO4

2.5 g sodium citrate?2H2O

1 liter of high quality distilled water

Once the ingredients are added, heat with stirring until the components are completely dissolved. Adjust to pH 7 with KOH. Pour the solution into smaller bottles with loosened caps and the autoclave at 15 lb/in2 for 15 min. If you wish to add add antibiotics or nutritional supplements, do this only after the autoclave cycle is complete, as the high temperature may destroy these components. Wait until the the bottle is less than 50°C (it should be warm to touch), and then add the components. After the bottles cool to below 40°C, the caps can be tightened and the concentrated medium stored indefinitely at room temperature.

Preparing the 1X Working Solution

To make 1X working solution, the 5X media should be diluted to 1× with high quality sterile distilled water.

Add the following sterile solutions

1 ml 1 M MgSO4.7H2O

10 ml 20% carbon source (sugar or glycerol)

Typically, several additional components are also added to make a complete medium.

Additional Components for 1X Working Solution

Procedure

Preparing the 5X stock

Add the following reagents to a 2-liter flask:

10 g (NH4)2SO4

68 g KH2PO4

2.5 mg FeSO4.7H2O

1 liter of high quality distilled water

Once the ingredients are added, heat with stirring until the components are completely dissolved. Adjust to pH 7 with KOH. Pour the solution into smaller bottles with loosened caps and the autoclave at 15 lb/in2 for 15 min. If you wish to add add antibiotics or nutritional supplements, do this only after the autoclave cycle is complete, as the high temperature may destroy these components. Wait until the the bottle is less than 50°C (it should be warm to touch), and then add the components. After the bottles cool to below 40°C, the caps can be tightened and the concentrated medium stored indefinitely at room temperature.

Preparing the 1X Working Solution

To make 1X working solution, the 5X media should be diluted to 1× with high quality sterile distilled water.

Add the following sterile solutions

1 ml 1 M MgSO4.7H2O

10 ml 20% carbon source (sugar or glycerol)

Typically, several additional components are also added to make a complete medium.

Additional Components for 1X Working Solution

Procedure

Preparing the 5X stock

Add the following reagents to a 2-liter flask:

30 g Na2HPO4

15 g KH2PO4

5 g NH4Cl

2.5 g NaCl

15 mg CaCl2 (optional)

1 liter of high quality distilled water

Once the ingredients are added, heat with stirring until the components are completely dissolved. Pour the solution into smaller bottles with loosened caps and the autoclave at 15 lb/in2 for 15 min. If you wish to add add antibiotics or nutritional supplements, do this only after the autoclave cycle is complete, as the high temperature may destroy these components. Wait until the the bottle is less than 50°C (it should be warm to touch), and then add the components. After the bottles cool to below 40°C, the caps can be tightened and the concentrated medium stored indefinitely at room temperature.

Preparing the 1X Working Solution

To make 1X working solution, the 5X media should be diluted to 1× with high quality sterile distilled water.

Add the following sterile solutions

1 ml 1 M MgSO4.7H2O

10 ml 20% carbon source (sugar or glycerol)

Typically, several additional components are also added to make a complete medium.

Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is often used as a selective agent in molecular biology to select for and to confirm the uptake of genes (e.g., of plasmids) by bacteria (e.g., E. coli). A gene that is to be inserted into a bacterium is coupled to a gene coding for an ampicillin resistance (in E. coli, usually the bla (TEM-1) gene, coding for beta-lactamase). The treated bacteria are then grown in a medium containing ampicillin (typically 50–100 mg/L). Only the bacteria that successfully take up the desired genes become ampicillin resistant, and therefore contain the other desired gene as well. As a powder, ampicillin is white with slight yellow cast and is soluble in water (150 mg/ml).